Topological defects in self-assembled patterns of mesenchymal stromal cells in vitro are predictive attributes of condensation and chondrogenesis

Mesenchymal stromal cells (MSCs) are promising therapeutic agents for cartilage regeneration, including the potential of cells to promote chondrogenesis in vivo. However, process development and regulatory approval of MSCs as cell therapy products benefit from facile in vitro approaches that can predict potency for a given production run. Current standard in vitro approaches include a 21 day 3D differentiation assay followed by quantification of cartilage matrix proteins. We propose a novel biophysical marker that is cell population-based and can be measured from in vitro monolayer culture of MSCs. We hypothesized that the self-assembly pattern that emerges from collective-cell behavior would predict chondrogenesis motivated by our observation that certain features in this pattern, namely, topological defects, corresponded to mesenchymal condensations. Indeed, we observed a strong predictive correlation between the degree-of-order of the pattern at day 9 of the monolayer culture and chondrogenic potential later estimated from in vitro 3D chondrogenic differentiation at day 21. These findings provide the rationale and the proof-of-concept for using self-assembly patterns to monitor chondrogenic commitment of cell populations. Such correlations across multiple MSC donors and production batches suggest that self-assembly patterns can be used as a candidate biophysical attribute to predict quality and efficacy for MSCs employed therapeutically for cartilage regeneration.


Introduction
The growing promise of cell therapy for tissue regeneration calls forth a need for rapid and label-free assays that can predict the potential of the cell product to differentiate into desired tissue type.The case considered here is that of mesenchymal stromal cell (MSC) therapy for cartilage repair.Since surgical and pharmaceutical methods of treating cartilage degeneration associated with osteoarthritis restore only limited function, mesenchymal stromal cells that have the potential to differentiate into chondrocytes are of potential translational interest.The approval of such cell-based products by health regulatory authorities anticipates demonstration of identity, safety, purity, and potency of the product.Assays are preferably rapid and label-free, enabling in-line qualification during the cell manufacturing process.However, the current standard in vitro assay for predicting cartilage regeneration potency of MSCs is chondrogenic differentiation, a relatively slow process comprising 21 days of differentiation in 3D pellet using 100K cells, followed by cartilage matrix protein quantification [1].So far, most biophysical models for predicting MSC differentiation have been based on single-cell features such as cell geometry, actin structure, nucleus geometry, etc. [2][3][4].However, MSC differentiation is a cell population-based phenomenon, as suggested by the high confluency requirement of the starting culture in MSC in vitro differentiation protocols [5].A recent study even suggests over-confluence of MSC expansion cultures for effective chondrogenesis, that is conventionally avoided as cell-cell contact inhibits growth [6].Accordingly, cell population-based features might offer a more direct prediction of the MSC differentiation outcome.
We hypothesized that the self-assembled patterns that emerge in confluent MSC monolayers would be predictive of the chondrogenic potential.This hypothesis was based on the rationale that certain features within the self-assembled pattern corresponded to mesenchymal condensations,-the self-aggregation of mesenchymal cells that is an early and critical step during the development of skeletal tissues [7][8][9].The mesenchymal condensations during skeletal development appear as a regularly-spaced pattern of spots that correspond to nodules of the developing limb.Conventionally, computational biologists have modelled the patterning of these condensations via chemotaxis and haptotaxis [10][11][12].
Interestingly, the self-assembled pattern of cellular swirls that emerges in cell monolayers has been extensively studied in the field of active matter physics, and modelled as kinetic phase transition [13], jamming transition [14], and glass transition [15].Recently, this swirl pattern has been compared to the turbulence pattern [16,17], and liquid crystal pattern [18,19].The liquid crystal-like pattern along with liquid crystal-like defects in cellular self-assembly has been observed in various biological systems [20,21] with discoveries that the so-called topological defect sites are the scenes for fundamental morphogenetic events, such as epithelial cell extrusion [22] and neural crest cell migration [23].In this work, we leverage the relationship between topological defects in cell monolayers and morphogenesis in tissue development to propose an assay and potential attribute predictive of the MSC cell therapy product potency for cartilage repair.
With the rationale that self-assembled patterning precedes morphogenesis, we hypothesized that the quantification of this pattern in confluent MSC monolayers would enable an early and straightforward prediction of chondrogenic potential.Through time-lapsed image analysis of the MSCs in expansion culture conditions (i.e., not chemically induced differentiation conditions) in two-dimensional multi-well formats, we observed a strong correlation between pattern variance at day 9 of cellular swirl assay with the protein level quantified from chondrogenic pellet after 3D chondrogenic differentiation assay at day 21.With development of advanced machine learning algorithms for forecasting the self-assembly patterns as well as in vivo validation, this candidate biophysical attribute can potentially be incorporated as a label-free critical quality attribute during cell manufacturing process.

Cellular swirls emerge in confluent cultures of bone marrow-derived MSCs (bm-MSCs)
A visual inspection of any in vitro culture on tissue culture plastic of bm-MSCs at confluency shows cellular swirls formed by alignment between neighboring cells (Fig 1A).To observe the origin of these emergent swirls, we performed time-lapse imaging for 2 weeks, starting from sparsely seeded single cells until confluency and emergence of swirls (S1 and S2 Movies).We observed that when local cell density reached a critical value, around day 10 in our movies, the cells transitioned into a collective motion resembling fluid-like behavior.This collective cell flow then produced turbulence-like vortices, building towards the appearance of swirls formed by alignment of neighboring cells.
Motivated by the possibility of using the cellular swirl pattern as a morphological feature for prediction of chondrogenic potential, we generated cellular swirls in bm-MSCs systematically by seeding cells in 48-well plates at a density of 50,000 cells/cm 2 for up to 12 days (Fig 1B , see Discussion for why this culture vessel and this cell density was chosen).To visualize the cellular swirls, we fixed the cells and stained their actin and nuclei using phalloidin and DAPI respectively because the 48-well plates were not suitable for phase-contrast imaging.Samples were fixed on day 3, 6, 9, or 12 post-seeding for the time study.Images of actin and nuclei were acquired for tiles across the whole well and stitched to capture the complete pattern formed in the well.Before proceeding with quantification of the swirl pattern, we qualitatively examined certain features in the pattern, especially the actin arrangement and cell density at centers of the swirls.

Cells self-aggregate at swirl centers, the so-called 'positive' topological defects
Zoomed-in actin images of the swirl pattern showed topological defects of 'comet' (+1/2) type and 'spiral' (+1) type.In the DAPI-stained nucleus images, these topological defect sites corresponded to highly dense cell clusters, often with overlapping nuclei (Fig 2 and S1 Fig).These high density cell clusters that appear at swirl centers resemble mesenchymal condensationsthe self-aggregation of mesenchymal cells during early stage of cartilage and bone development [7][8][9].The reverse was observed at topological defects of the 'tri-radius' (-1/2) type which corresponded to local minima of cell density in the nucleus image (S2 Fig).

Self-aggregated cell clusters correspond to mesenchymal condensations
To test whether the cells that aggregate at +1/2 and +1 defect sites indeed correspond to mesenchymal condensations, we stained the day 8 samples with fluorescently tagged peanut agglutinin (PNA) antibody, a lectin binding protein used for labelling mesenchymal condensations [24].Zoom-in images show high intensity of PNA at the cell clusters (Fig 3).Further, immunostaining of transcription factors YAP, SOX9, and RUNX2 showed high intensity in the nuclei within the cell aggregates (S3 Fig).

Swirl pattern is cell batch-specific and reproducible
We performed a qualitative comparison of the swirl patterns across the phase contrast images of confluent MSCs collected from cultures of various bm-MSC donors maintained by different researchers in our lab.We observed qualitative similarities in the patterns made by the same donor across images captured by different researchers in different months (of 80-90% confluent culture on day 10-12 post seeding with an initial density of 1500-2000 cells/cm 2 ) (S4 Fig).whether a quantification of this swirl pattern across various donors correlates with their in vitro chondrogenic differentiation potential, since cellular swirls lead to mesenchymal condensations, a crucial step that precedes chondrogenic differentiation.

Swirl pattern quantification correlates with in vitro chondrogenic differentiation potential
To find an image-based early marker of chondrogenic differentiation potential in MSCs, first, the chondrogenic differentiation assay was conducted for 5 donors (see Methods).Briefly, this assay involved 3 week 3D pellet culture in chondrogenic differentiation condition, followed by quantification of cartilage matrix proteins sulfated glycosaminoglycans (sGAGs) and collagen-II (Col2).Simultaneously, for generating cellular swirls, cells from the same 5 donors were cultured at 50,000 cells/cm 2 in 48-well plates and stained for nucleus and actin at days 3,6,9, and 12 post seeding.For image quantification, two different methods were adopted; one using nucleus image to directly quantify the mesenchymal condensations, and the other using actin image to indirectly quantify the condensations as topological defects in the swirl pattern.In the first method, the total area of cell clusters was quantified, for which the nucleus images were thresholded, and the total area of segmented regions was measured (S5A and S5B Fig, see Methods).While the total area of clusters per well increased from day 3 to day 12 for all donors (S5C Fig), it did not correlate strongly with the chondrogenic differentiation quantified using cartilage matrix protein expression (Pearson's correlation coefficient r < 0.5).In the second method, swirl pattern was quantified from actin orientation, as we had observed that topological defects in swirl pattern correspond to mesenchymal condensations (Fig 3)-an early marker of chondrogenic differentiation.Hence, we explored a quantification method that would differentiate the actin organization at topological defect from the other regions having fully ordered or disordered actin.For the purpose of illustration, we cropped from whole-well actin images, three small regions corresponding to each type of pattern: 'topological defect', 'disorder', and 'order' (Fig 5A).The correspondingly cropped nucleus images (S6 Fig) confirmed occurrence of condensation only for the topological defect region.Next, the orientation and coherency images were generated from the actin images for the three regions using the open source plugin OrientationJ for Fiji (see Methods).The orientation shows the direction of local features, and varies from -90 to +90 degrees.The coherency measures the local structure: it is 1 when the local structure has a dominant orientation and 0 when the local structure is unaligned or isotropic.Subsequent computation of the variance of coherency (VoC) for the three regions showed that the topological defect region has higher VoC compared to regions that are disordered or highly ordered.
Following this method, VoC was computed from all whole-well actin images (5 donors x 4 time points x 3 technical replicates = 60 whole-well actin images) (see Methods).The quantity VoC showed high technical reproducibility as suggested by a low coefficient of variation (less than 30%) for all donors (S7 Fig) .Next, we tested the correlation of VoC with cartilage matrix protein quantification across 5 donors at the 4 time points: day 3, 6, 9, and 12. Interestingly, the VoC at day 9 showed strong correlation with both cartilage matrix components, generating a Pearson's correlation coefficient r = 0.88 for sGAG, and r = 0.98 for Col2 (Fig 5B ), while the VoC at earlier time points did not correlate strongly with the differentiation markers (S8 Fig) .Next, we tested whether the quantity VoC could be measured from phase-contrast images of living (unfixed) cells, as this would have implications for in-line label-free monitoring of chondrogenic differentiation efficiency.We observed that the coherency histogram obtained from actin image matches that obtained from phase-contrast image for the same region (S9 Fig).

Discussion
The use of mesenchymal stromal cell therapy for cartilage repair requires an assessment of the efficacy of the MSC product to repair the cartilage tissue.The current in vitro assay that assesses the cartilage formation efficacy of MSCs is the chondrogenic differentiation assay comprising of 21 days of 3D pellet culture followed by quantification of cartilage matrix proteins.There is a need for efficacy prediction assays that enable early prediction and are nondestructive, so they can be incorporated as in-line assays within the cell manufacturing pipeline.Our work provides proof-of-concept that the pattern of self-assembly in mesenchymal stromal cells can predict their chondrogenic potential in 9 days with scope for an even earlier and label-free prediction.
We hypothesized that cellular swirl patterns emerging on tissue culture plastic from collective self-organization of cells would be predictive of the chondrogenic differentiation potential of the batch of cells since such self-organization is known to precede tissue morphogenesis.To test this, we first generated the self-assembled cellular swirls in confluent human bone marrow-derived MSCs by controlling seeding density, culture vessel geometry, and culture duration.The value of starting cell density was chosen such that cells are close to 90% confluent upon attachment, as this would minimize the time required for emergence of cellular swirls.The culture vessel chosen was 48-well plate, so as to optimize between the number of swirl features per well and the byte-size of the whole-well overview image.Recent studies of collective cell behavior in field of active matter physics suggest that the patterning observed in biological tissues resembles the patterns and topological defects observed in nematic liquid crystals [18,19].A salient feature of the nematic liquid crystal pattern is topological defects, i.e. the sites within the pattern where sudden changes in orientation occur, for example, the centers of the spiral swirls [25].We were able to identify positive and negative, integer and halfinteger types of topological defects within the patterns visible in actin-stained images of the cellular swirls.We observed self-aggregated cell clusters located at the so-called 'comet' (+1/2) and 'spiral' (+1) topological defect sites within the nematic pattern.Such clustering of particles at positively charged topological defect sites can be induced by turbulence-like flow in active systems as is recently being explored [26].These cell clusters were identified as mesenchymal condensations, the early stage of cartilage formation, via labelling with peanut agglutinin antibody.Further, we observed high expression of transcription factors YAP, SOX9, and RUNX2 in the cells within the clusters.This suggests that the high mechanical stresses, known to arise at +1/2 and +1 defect sites, possibly activate the mechanosensitive transcription factor YAP in the mesenchymal condensations, followed by downstream activation of RUNX2 and SOX9, the master transcription factors of osteogenic and chondrogenic differentiation [27].
Interestingly, we observed that the swirl patterns made by cells obtained from different donors were cell batch-specific and reproducible across technical replicates.Next, we explored the possibility of using swirl pattern to predict the chondrogenic differentiation potential of MSCs.Toward this, we adopted two methods of pattern quantification-using nucleus image to quantify the total area of mesenchymal condensations or cell clusters, and using the actin image to quantify the topological defects.The first quantity, i.e. total area of cell clusters computed from nucleus image did not correlate strongly with chondrogenic differentiation markers.This could have two possible implications: (1) higher total area of clusters does not necessarily mean better chondrogenesis, or (2) the total area computed from intensity-based thresholding does not successfully capture the quality of mesenchymal condensations.The second quantity, computed from actin images, captured the level of order in the actin orientation.This quantity, 'coherency', ranges from 0 to 1.It is uniformly high in regions that have fully ordered actin fiber arrangement, uniformly low in regions that have for fully disordered arrangement, and is spread from low to high in regions that have a topological defect.Thus, we computed the 'variance of coherency' (VoC) as a measure of the topological defects in the actin image.The VoC is high for regions corresponding to defect and low for fully ordered or fully disordered regions.Interestingly, the VoC of day 9 swirl pattern correlated strongly with the cartilage matrix proteins expressed on day 21 of the chondrogenic differentiation assay (n = 5 donors).The low correlation of VoC at earlier time points (VoC of day 3 and day 6) with the differentiation markers is possibly because of lag in emergence of cellular swirls across donors, since differences in cell morphology and proliferation rates would affect the value of critical density at which the swirls emerge and the time required to achieve that critical density.To summarize, we showed that swirl-pattern quantification could be used as a potential critical quality attribute for predicting efficacy of mesenchymal stromal cell product for cartilage repair.Here, we do not claim the patterns to be donor-specific, or inherently genetic, but rather cell preparation condition-specific.We can refer to this as batch specific.This distinction is important because MSC phenotype is established to be easily moderated by culture conditions, and a change in those conditions could alter both the observed VoC and chondrogenic potential of that cell batch.Further aspects regarding implications of swirl-pattern quantification for cell therapy manufacturing are discussed below.
A crucial stage during the cell therapy manufacturing is that of cell expansion.Typical protocol optimized for expansion recommends seeding mesenchymal stromal cells sparsely and harvesting them before they reach confluency [28,29].Hence, a prediction model based on single-cell morphological features could be easily incorporated in-line within the manufacturing pipeline.While cellular swirls only appear at high confluency, advances in forecasting of active nematics [30] from morphology, migration rate, and proliferation rate of single-cells could pave the way for an earlier prediction from lower confluency of ~80%, typical of the MSC harvest stage of the cell therapy manufacturing.Secondly, with the new understanding that cellular swirls lead to mesenchymal condensations, cell manufacturers for cartilage regeneration could consider culturing MSCs to near 100% confluency to prime the cells towards chondrogenesis.In such a case, an in-line label-free monitoring of chondrogenic differentiation efficacy via swirl pattern would be feasible, as we have shown that VoC can also be measured from phase-contrast images of living (unfixed) cells.Thirdly, culturing MSCs on engineered patterned surfaces that induce formation of topological defects [31] would provide a better control for manufacturing desired cell therapy product.
Growing evidence from in vivo cartilage repair studies shows that the implanted MSCs play an indirect role in tissue repair via secretion of paracrine factors as opposed to directly differentiating into chondrocytes [32].While our current work shows that collective cell patterns provide an early prediction of the in vitro chondrogenic differentiation potential, future studies can establish whether these patterns also predict the in vivo cartilage repair outcome.

Cell-source and cell expansion
Human adult bone marrow-derived MSCs (bm-MSCs) from 5 different donors (males, age 20-30 years) were purchased from Lonza and StemCell Technology.The first round of expansion for all donors from passage 1 to passage 2 was done by a single researcher.Cells were seeded in culture flasks (ThermoFisher Nunc) at 2000 cells/cm 2 using growth medium comprising of Dulbecco's modified Eagle's medium, low glucose and pyruvate (Gibco), supplemented with 10% MSC certified fetal bovine serum (Gibco) and 1% penicillin and streptomycin (Gibco).Medium was changed every 3 days until the cells reached ~80% confluency, typically 1-2 weeks, when the cells were harvested using the trypsin (Gibco).The second passage cells were then stored in common repository by cryo-preserving in batches of 3.5*10 5 per vial in freezing medium comprising of growth medium with 10% DMSO (Sigma).For subsequent use in various experiments, different researchers typically expanded the MSCs from passage 2 to passage 3 or passage 4 following the same expansion protocol as above.For generating cellular swirls in 48-well plates in the current study, second passage vials of all 5 donors were first thawed, and serial expansion was performed until a cell-count of 7*10 5 cells was achieved for each donor (~0.6*10 5 cells/well x 3 technical replicates x 4 time-points).Typically, this cell count was achieved at the third passage for fast-growing donors and fourth passage for slow-growing donors.

Sample preparation for generating cellular swirls
Cells were seeded in 48-well plates at a density of 50,000 cells /cm 2 .Note that the first and last rows as well as the first and last columns of the well-plate were not used for seeding because limitations in XY translation of the microscope stage affects the whole-well imaging for these wells.Also, samples corresponding to different time-points were seeded in different well-plates for the reason that paraformaldehyde (PFA) vapors from the samples being fixed resulted in cell death in other samples within the same well plate.For the time-study experiments, the samples were fixed on either day 3, 6, 9, or 12 with medium change every 3 days until they're fixed.For fixing, growth medium was replaced with 4% PFA in PBS (Biotium, 200ul per well) for 20 minutes, following which the PFA was replaced with PBS.The fixed samples were stored in the incubator or refrigerator until all time points were fixed and ready for staining.

Fluorescent staining
Fixed samples were first permeabilized using 0.1% triton X-100 detergent solution (Thermo Scientific) for 5 minutes, followed either by blocking and primary antibody staining or directly by 30 minute staining of nucleus (NucBlue Fixed Cell ReadyProbes, Life Technologies) and actin (ActinGreen 488 ReadyProbes, Life Technologies).

Sulfated glycosaminoglycan (sGAG) and type II collagen quantification
Samples were digested with 10 mg/mL of pepsin in 0.05 M acetic acid at 4˚C, followed by digestion with elastase (1 mg/mL).The amount of sulfated glycosaminoglycan (sGAG) was quantified using Blyscan sGAG assay kit (Biocolor, UK) according to manufacturer's protocol.Absorbance was measured at 656 nm and sGAG concentration (μg per μg DNA) was extrapolated from a standard curve generated using a sGAG standard.Type II Collagen (Col 2) content was measured using a captured enzyme-linked immunosorbent assay (Chondrex, Redmond, WA).Absorbance at 490 nm was measured and the concentration of Col 2 (ng per μg DNA) was extrapolated from a standard curve generated using a Col 2 standard.The amount of both sGAG and Col 2 content were normalized to the DNA content of respective samples, measured using Picogreen dsDNA assay (Molecular Probes, OR, USA).Three replicates were analyzed within each group.

Fig 1 .
Fig 1. Cellular swirls emerge in confluent cultures of mesenchymal stromal cells.(A) Left panel shows phase-contrast image of bone marrow-derived mesenchymal stromal cells cultured to confluency in T75 flasks.Right panel shows its corresponding orientation image generated using OrientationJ plugin in ImageJ.(B) Experiment methodology showing cell sample preparation, staining, imaging, and image processing to generate whole well pattern images.All scale bars are 1000 μm.https://doi.org/10.1371/journal.pone.0297769.g001

Fig 2 .
Fig 2. Cells aggregate at +1/2 and +1 topological defect sites.Six regions (cropped from whole-well stitched images) showing that cells aggregate at sites of +1/2 and +1 topological defects.The aggregates are visible in the nucleus images, while the defects are visible in the actin and corresponding orientation vectorfield images.Orientation vectorfields were generated using the OrientationJ plugin in ImageJ (see Methods).Scale bar 1000 μm.https://doi.org/10.1371/journal.pone.0297769.g002

Fig 3 .
Fig 3. Cell aggregates at topological defects correspond to mesenchymal condensations.The cell aggregates arising at defect sites of actin nematic pattern colocalize with peanut agglutinin (PNA), the marker for mesenchymal condensations-a pre-requisite for formation of cartilage or bone during skeletal development.Scale bar 1000 μm.https://doi.org/10.1371/journal.pone.0297769.g003

Fig 4 .
Fig 4. Self-assembled swirl pattern is cell batch-specific.Whole-well actin images of cellular swirls generated from two cell sources on day 3 post-seeding.The small-panels show zoomed-in regions cropped from the whole-well actin pattern.Cell source A = donor 1 passage 3, cell source B = donor 2 passage 3. Cells were seeded at 50,000 cells / cm2 for cell sources.Samples for both cell sources were fixed and stained on day 3 post seeding.Scale bar 1000 μm.https://doi.org/10.1371/journal.pone.0297769.g004

Fig 5 .
Fig 5. Variance of pattern coherency correlates with in vitro chondrogenic differentiation.(A) The first row shows actin images corresponding to three regions where the pattern may be classified as having 'disorder', 'order', and 'topological defect'.The second and the third rows show orientation and coherency images for the three regions, generated from the corresponding actin images using OrientationJ (see Methods).The values of orientation vary from -90 to +90 degrees, and the values of coherency vary from 0 to 1.The last row shows histogram of coherency images.
Scalebar 1000 μm.(B) XY scatter plots, where X = variance of coherency (VoC) calculated from day 9 patterns; and Y = expression level of cartilage matrix proteins sGAG and Col2 quantified from the 21 day chondrogenesis differentiation assay.The dots correspond to mean of 3 technical replicates, while the X and Y error bars correspond to standard error.Red line shows the linear fitting.https://doi.org/10.1371/journal.pone.0297769.g005